Behavioral effects of inhibition of cannabinoid metabolism: The amidase inhibitor AM374 enhances the suppression of lever pressing produced by exogenously administered anandamide.

Biochemical investigations have identified putative enzymatic pathways for the synthesis and metabolism of endogenous cannabinoids. Anandamide amidase is an enzyme that metabolizes anandamide into arachadonic acid and ethanolamine. Using in vitro methods, various inhibitors of amidase have been identified. The present studies were undertaken to determine if the amidase inhibitor AM 374 could enhance the effects of intraperitoneal (IP) injections of anandamide. Three studies were conducted to investigate the effects of various drug treatments on fixed ratio 5 operant lever pressing for food reinforcement. In the first study, the effects of different doses of anandamide were assessed, and it was demonstrated that 5.0 and 10.0 mg/kg anandamide IP significantly suppressed lever pressing, while 2.5 mg/kg produced very little effect. The second study tested the effects of intraventricular (ICV) injections of AM 374, and it was observed that doses up to 10.0, 20.0 and 40 microg AM 374 had no significant effect upon lever pressing. The third study investigated the combined effect of AM374 with a low dose of anandamide. Rats received two drug injections: one ICV and one IP. Four different drug treatments were assessed: 1) ICV vehicle + IP vehicle, 2) ICV vehicle + 2.5 mg/kg anandamide IP, 3) ICV 20.0 microg AM 374 + IP vehicle, and 4) ICV 20 microg AM 374 + 2.5 mg/kg anandamide IP. Combined administration of AM 374 plus anandamide led to a significant decrease in lever pressing compared to either AM374 or anandamide administered alone. Observations of the animals treated with the combination of AM374 plus anandamide indicated that the drug combination resulted in motor slowing, which is consistent with the notion that stimulation of cannabinoid receptors produced a motor deficit that interfered with lever pressing. Although AM374 produced no effect on its own, this amidase inhibitor did enhance the behavioral effect of a low dose of anandamide. These results are consistent with the notion that AM 374 inhibited the enzymatic breakdown of exogenously injected anandamide. This type of procedure can be used to assess a variety of different compounds for their ability to inhibit cannabinoid metabolism.

The cannabinoid CB1 antagonists SR 141716A and AM 251 suppress food intake and food-reinforced behavior in a variety of tasks in rats.

Cannabinoid CB1 receptor agonists, including delta-9-tetrahydrocannabinol (Delta 9-THC) (the main psychoactive ingredient in marijuana) have been shown to increase feeding in rats and humans. Conversely, it has been reported that acute administration of the CB1 receptor antagonist SR 141716A reduces food intake in rats. Based upon this observation, it has been suggested that CB1 antagonists could be useful as appetite suppressant drugs. The present studies were designed to provide a detailed examination of the effects of CB1 antagonists on food intake across a range of paradigms. Two CB1 antagonists (SR 141716A and AM 251) were administered to rats trained on fixed-ratio schedules with two different ratio requirements (fixed-ratio 1 and fixed-ratio 5). Both drugs produced a dose-dependent decrease in lever pressing, and had a relatively long duration of action (T1/2: SR 141716A, 15.1 h; AM 251, 22.0 h). Furthermore, intake of three diets with differing macronutrient composition (lab chow, high fat, high carbohydrate) was studied. Both drugs significantly suppressed intake of all three foods, and there were no significant interactions between drug dose and diet type. These findings support the hypothesis that CB1 receptor antagonists could be useful pharmacological tools for the suppression of appetite.

Dopamine antagonists alter response allocation but do not suppress appetite for food in rats: contrast between the effects of SKF 83566, raclopride, and fenfluramine on a concurrent choice task.

RATIONALE: Dopamine is important for enabling organisms to overcome work-related response costs. One way of investigating this function has been with concurrent choice procedures using food reinforcement. In the present study, rats were given a choice between pressing a lever for preferred Bioserve pellets, or approaching and consuming a less-preferred laboratory chow that was concurrently available. In previous work with this task, dopamine antagonists and accumbens dopamine depletions decreased lever pressing but increased chow consumption. OBJECTIVE: The present study assessed three drugs (two dopamine antagonists and one appetite suppressant) using the lever pressing/chow feeding task. RESULTS: Under baseline conditions, rats pressed the lever at high rates (1,300-1,500 responses) to obtain the preferred food, and little of the laboratory chow was eaten (1-2 g). Selective D1 and D2 antagonists (SKF 83566 and raclopride) reduced fixed ratio 5 lever pressing, but substantially increased chow consumption. In contrast, the serotonergic appetite suppressant fenfluramine reduced both lever pressing and chow consumption. With the dopamine antagonists, lever pressing and chow consumption were inversely correlated across treatments, while these two measures were unrelated in the fenfluramine experiment. CONCLUSIONS: Dopamine antagonists and accumbens dopamine depletions do not simply reduce appetite. Rats with accumbens dopamine depletions, or rats treated with low doses of selective or non-selective dopamine antagonists, remain directed toward the acquisition and consumption of food. These results demonstrate that fundamental aspects of food reinforcement are left intact after treatment with low doses of dopamine antagonists.

Nucleus accumbens dopamine depletions make rats more sensitive to high ratio requirements but do not impair primary food reinforcement.

It has been suggested that nucleus accumbens dopamine is involved in the process of enabling organisms to overcome work-related response costs. One way of controlling work requirements in operant schedules is to use fixed ratio schedules with different ratio requirements. In the present study, the effects of nucleus accumbens dopamine depletions were assessed using four schedules: fixed ratio 1, 4, 16, and 64. Rats with nucleus accumbens dopamine depletions showed behavioral deficits that were highly dependent upon the ratio value; there were no effects of dopamine depletions on fixed ratio 1 lever pressing, but as ratio value got larger, the impairment became much greater. In a separate experiment, pre-feeding to reduce food motivation was shown to produce a different pattern, such that performance on all ratio schedules was substantially impaired. Thus, aspects of food reinforcement that are involved in fixed ratio 1 performance are highly sensitive to food motivation, but are largely unaffected by nucleus accumbens dopamine depletions. Nevertheless, nucleus accumbens dopamine depletions affected the elasticity of demand for food, and enhanced "ratio strain", i.e. they exacerbated the response-suppressing effects of increasing ratio value.

Effects of dopamine antagonists and accumbens dopamine depletions on time-constrained progressive-ratio performance.

Four experiments were conducted to determine the effects of dopamine (DA) antagonists and DA depletions on progressive-ratio responding for food reinforcement. On this schedule, ratio requirement increased by one response after each reinforcer was obtained, and rats were tested in 30-min sessions. Response rates and highest ratio completed were reduced in a dose-related manner by systemic injections of the D1 antagonist SCH 23390, and also by the D2 antagonists haloperidol and raclopride. Drug-treated rats also showed reductions in time to complete the last ratio, demonstrating that they had stopped responding before the end of the session. DA depletions produced by injections of 6-OHDA directly into the nucleus accumbens substantially decreased both the number of responses and the highest ratio completed. The deficits in response number and highest ratio completed induced by DA depletions persisted through the first 3 weeks of postsurgical testing, with some recovery by the fourth week. However, the deficits resulting from dopamine depletions were largely a manifestation of a decrease in response rate; although time to complete the last ratio was significantly reduced by dopamine depletions in the first few days of testing, rats recovered on this measure by the fifth day after surgery. Although previous work has shown that performance on several schedules (e.g., continuous, low value ratios, variable interval) is relatively unaffected by accumbens DA depletions, the present data demonstrate that such depletions do produce a substantial and persistent impairment of progressive ratio response output. Rats with accumbens DA depletions appear to have deficits in maintaining the high work output necessary for responding at large ratio values. The relative sparing of responding on some simple schedules, together with the present progressive ratio results, suggest that rats with accumbens DA depletions remain directed toward the acquisition and consumption of food, but they show deficits in work output for food.

A detailed characterization of the effects of four cannabinoid agonists on operant lever pressing.

The present experiments were conducted to investigate the effects of four cannabimimetics on detailed temporal parameters of operant responding. In this study, the behavioral output during performance of a fixed ratio 5 schedule of reinforcement was recorded by a computer program that measured the response initiation time (IT; time interval between the offset of one lever press and the onset of the next) and the response duration (the amount of time that elapses from the onset to the offset of one lever press) of each lever press. ITs were further partitioned into fast responses (IT=0.0-1.0 s), short pauses (IT= 1.0-2.5 s), and long pauses (IT>2.5 s). Four cannabimimetic agents were assessed in this study: (R)-methanandamide (AM 356), a hydrolytically stable analog of arachidonylethanolamide, an endogenous ligand for the CB1 receptor; CP-55,940, a potent non-classical synthetic ligand; (-)-delta8-tetrahydrocannabinol (delta8-THC), an isomer of the naturally occurring delta9-THC; and WIN 55,212-2, a synthetic aminoalkylindole. All four of the cannabimimetic drugs tested significantly suppressed operant lever pressing in a dose dependent manner. The rank order of potencies observed in the present study was CP-55,940>>WIN-55,212-2>delta8-THC>AM 356, which is consistent with the rank order of affinities for the CB1 receptor shown by these drugs. All of the cannabimimetics substantially increased average IT, and also increased duration time. There was a substantial increase in average length of long pauses, and statistically significant but very small changes in the local rate of responding as measured by the average length of fast ITs. Cannabinoid-treated rats were largely immobile during pauses in responding, and these animals showed several signs of ataxia and catalepsy at the doses that suppressed lever pressing. Together with other data, the present results suggest that CB1 stimulation leads to motor effects that are associated with a suppression of lever pressing.

Effects of subchronic administration of clozapine, thioridazine and haloperidol on tests related to extrapyramidal motor function in the rat.

Clozapine, thioridazine (THIO) and haloperidol were administered for 14 consecutive days, and separate groups of rats were used to study the effects of these drugs on tremulous jaw movements and lever pressing. Rats were observed on day 13 for the ability of the antipsychotic drugs to induce jaw movements. Haloperidol produced a dose-related increase in jaw movements, while clozapine and THIO failed to induce jaw movements. On day 14, rats were challenged with 5.0 mg/kg of the anticholinesterase tacrine, which induces a very high level of jaw movement activity. Clozapine significantly reduced tacrine-induced tremulous jaw movements, while haloperidol did not. Although previous work had shown that acute THIO could suppress jaw movements, repeated THIO failed to do so. In order to provide an additional behavioral test for comparisons of the relative potencies of the antipsychotic drugs, rats were tested for the effects of these drugs on fixed ratio 5 lever pressing. All three drugs significantly suppressed lever pressing. Haloperidol showed sensitization with repeated injections, while clozapine showed tolerance. Data were analyzed by taking the ratio of the ED50 for suppression of tacrine-induced jaw movement over the ED50 for suppression of lever pressing on day 14. Clozapine reduced tacrine-induced jaw movements in a dose range slightly lower than that required for reduction of lever pressing. In contrast, THIO and haloperidol failed to affect tacrine-induced jaw movements even at doses that were 5-18 times the ED50 for suppression of lever pressing. Thus, tests of jaw movement activity and lever pressing after repeated administration may be useful for assessing atypical antipsychotic drugs.

Effects of clozapine, thioridazine, risperidone and haloperidol on behavioral tests related to extrapyramidal motor function.

Evidence indicates that the antipsychotic drug clozapine has a low propensity for the induction of extrapyramidal motor symptoms, and also that clozapine has therapeutic effects in patients with idiopathic Parkinson's disease. Because tacrine-induced tremulous jaw movements in rats have been suggested as a possible model of extrapyramidal motor dysfunctions, including parkinsonian tremor, the present work was undertaken to investigate the effects of clozapine on tremulous jaw movements. Clozapine decreased tacrine-induced tremulous jaw movements in a dose-related manner, with an ED50 of approximately 3.3 mg/kg. In order to determine the relative potency of this effect compared to other behavioral effects of clozapine, suppression of lever pressing was also studied. Clozapine reduced lever pressing in a dose-related manner, with an ED50 of approximately 5.4 mg/kg. This indicates that clozapine suppressed jaw movements at or below the doses required for suppression of lever pressing. In contrast, the typical antipsychotic drug haloperidol failed to suppress tacrine-induced tremulous jaw movements in doses up to 1.0 mg/kg, which is about 11-fold higher than the ED50 for suppression of lever pressing with that drug. Thioridazine and risperidone also suppressed tremulous jaw movements in roughly the same dose range at which lever pressing was reduced. It is possible that the suppression of tacrine-induced tremulous jaw movements by clozapine in rats is related to the unique behavioral and motor effects of clozapine. The ratio of potencies of these effects (i.e., suppression of tremulous jaw movements versus suppression of lever pressing) could be used as a behavioral procedure for assessing clozapine-like activity in novel compounds.

Motor dysfunction produced by tacrine administration in rats.

In the present study, three experiments were conducted to provide a characterization of some of the motor effects of the anticholinesterase tacrine (1.25-5.0 mg/kg I.P.) in rats. In the first experiment, tacrine was found to produce tremulous jaw movements in the dose range of 1.25-5.0 mg/kg. The second experiment examined the effects of tacrine on locomotion, and it was demonstrated that tacrine produced a dose-related suppression of open-field motor activity. In the third experiment, the effects of tacrine were assessed using operant conditioning procedures. Behavioral output during lever pressing on a fixed ratio 5 schedule was recorded by a computerized system that measured response initiation time (time from offset of one response to onset of the next) and duration for each lever press. Tacrine administration substantially depressed lever pressing response rate. This deficit was largely due to a substantial increase in the average response initiation time. Analysis of the distribution of response initiation times indicated that tacrine-treated rats made relatively few responses with fast initiation times (e.g., 0-125 ms), and also that tacrine led to a dramatic increase in the number of pauses in responding (i.e., response initiation times greater than 2.5 s). Tacrine-treated rats showed a slight increase in the average initiation time for fast responses (i.e., a slight decrease in the local rate of responding), and also showed a substantial increase in the average length of pauses greater than 2.5 s. Analysis of response durations indicated that there was an overall increase in average response duration among animals that received the higher doses of tacrine. Although tacrine-induced decreases in the local rate of responding and increases in response duration contribute to the overall deficit, the major reason why tacrine-treated animals responded less was because they took much longer breaks in responding. It is possible that the tacrine-induced increases in pausing reflect a drug-induced akinesia. Thus, the present experiments indicate that tacrine impairs several aspects of motor function in the dose range tested. In view of the fact that tremor and motor slowing are classic symptoms of Parkinsonism, the present results in rats are consistent with the human literature indicating that tacrine (Cognex) can produce Parkinsonian side effects. Studies of the motor dysfunctions produced by tacrine in rats could be useful for investigating the motor side effects of tacrine in humans.